Bottom Line:
In this study, we used the bioorthogonal chemical reporter strategy (BOCR) in combination with label-free quantitative mass spectrometry (LFQ-MS) to characterize and accurately quantify the individual cell surface sialoproteome in human GBM tissues, in fetal, adult human astrocytes, and in human neural progenitor cells (NPCs).We identified and quantified a total of 843 proteins, including 801 glycoproteins.Among the 843 proteins, 606 (72%) are known cell surface or secreted glycoproteins, including 156 CD-antigens, all major classes of cell surface receptor proteins, transporters, and adhesion proteins.

ABSTRACTGlioblastoma multiform (GBM) remains clinical indication with significant "unmet medical need". Innovative new therapy to eliminate residual tumor cells and prevent tumor recurrences is critically needed for this deadly disease. A major challenge of GBM research has been the identification of novel molecular therapeutic targets and accurate diagnostic/prognostic biomarkers. Many of the current clinical therapeutic targets of immunotoxins and ligand-directed toxins for high-grade glioma (HGG) cells are surface sialylated glycoproteins. Therefore, methods that systematically and quantitatively analyze cell surface sialoglycoproteins in human clinical tumor samples would be useful for the identification of potential diagnostic markers and therapeutic targets for malignant gliomas. In this study, we used the bioorthogonal chemical reporter strategy (BOCR) in combination with label-free quantitative mass spectrometry (LFQ-MS) to characterize and accurately quantify the individual cell surface sialoproteome in human GBM tissues, in fetal, adult human astrocytes, and in human neural progenitor cells (NPCs). We identified and quantified a total of 843 proteins, including 801 glycoproteins. Among the 843 proteins, 606 (72%) are known cell surface or secreted glycoproteins, including 156 CD-antigens, all major classes of cell surface receptor proteins, transporters, and adhesion proteins. Our findings identified several known as well as new cell surface antigens whose expression is predominantly restricted to human GBM tumors as confirmed by microarray transcription profiling, quantitative RT-PCR and immunohistochemical staining. This report presents the comprehensive identification of new biomarkers and therapeutic targets for the treatment of malignant gliomas using quantitative sialoglycoproteomics with clinically relevant, patient derived primary glioma cells.

Mentions:
For a subset of 23 genes, the proteomic and microarray results were further validated by quantitative TaqMan real-time PCR assays for gene expression in GBM patient tumors and adult astrocytes. Of these 23 genes, 21 encode for proteins overexpressed (Effect size >3) in all four primary cultures derived from GBM patient tumors compared to human astrocytes and NPCs. The two remaining genes encode for EGFR and TFRC, two sialoglycoproteins equally expressed in the tumor group and the astrocyte group (Table S1). Expression data for individual genes are presented in Table S6. These data are also displayed in a heat map to highlight the broad similarities in gene expression that exist between tumor cells derived from 3 GBM patients relative to adult astrocytes (Fig. 7A). For 19 of the 23 selected genes, we found a significant positive correlation between mRNA expression and protein expression differences seen between GBM cells and astrocytes, indicating that a substantial proportion of protein changes between tumor cells and astrocytes were a consequence of changed mRNA levels (Table 3 and Fig. 7B). Of the 23 proteins, only four (ADCY6, LGALS3BP, THBD, and TTYH3) with upward trends had an opposite mRNA trend, these discrepancies might be the result of the post-transcription and/or post-translation regulation. Of the 23 selected genes, qRT-PCR results were consistent with the microarray results for 16 of the 19 genes whose mRNA levels were analyzed with both methods (ADCY6, AGT, ANGPTL2, CD68, CLU, CRYAB, EGFR, TFRC, GPR56, IL13RA2, ILRAP, ITGB8, NTRK2, PTPRZ1, SLC12A7 and SLC1A3). For three genes (CLCN3, THBD and TNC), microarray and qRT-PCR analyses gave inconsistent results. Overall, our results show a statistically significant correlation between protein and mRNA expression levels for the cell surface proteins up-regulated in GBM tumors. We clearly confirm overexpression of 25 cell surface proteins in GBM tumors compared to astrocytes and NPCs, as the result from microarray and qRT-PCR analyses were overall in accordance with those of our differentially expressed glycoproteomic analysis. Thus, the genes showing the highest expression levels in GBM tumors compared to astrocytes, at both mRNA and protein levels, include SLC1A3, PTPRZ1, GPR56, NTRK2, IL13RA2, NRCAM, AGT and CLU. It’s interesting to note that our proteome and transcriptome results also indicated that the expression of EGFR, the most commonly altered gene in GBM with overexpression and/or mutation occurring in at least 50% of cases [46], was not significantly different between the tumor group and the astrocyte group. Our results are consistent with previous findings showing a loss of EGFR amplification in primary cultured gliomas [34], [35], [47], [48].

Mentions:
For a subset of 23 genes, the proteomic and microarray results were further validated by quantitative TaqMan real-time PCR assays for gene expression in GBM patient tumors and adult astrocytes. Of these 23 genes, 21 encode for proteins overexpressed (Effect size >3) in all four primary cultures derived from GBM patient tumors compared to human astrocytes and NPCs. The two remaining genes encode for EGFR and TFRC, two sialoglycoproteins equally expressed in the tumor group and the astrocyte group (Table S1). Expression data for individual genes are presented in Table S6. These data are also displayed in a heat map to highlight the broad similarities in gene expression that exist between tumor cells derived from 3 GBM patients relative to adult astrocytes (Fig. 7A). For 19 of the 23 selected genes, we found a significant positive correlation between mRNA expression and protein expression differences seen between GBM cells and astrocytes, indicating that a substantial proportion of protein changes between tumor cells and astrocytes were a consequence of changed mRNA levels (Table 3 and Fig. 7B). Of the 23 proteins, only four (ADCY6, LGALS3BP, THBD, and TTYH3) with upward trends had an opposite mRNA trend, these discrepancies might be the result of the post-transcription and/or post-translation regulation. Of the 23 selected genes, qRT-PCR results were consistent with the microarray results for 16 of the 19 genes whose mRNA levels were analyzed with both methods (ADCY6, AGT, ANGPTL2, CD68, CLU, CRYAB, EGFR, TFRC, GPR56, IL13RA2, ILRAP, ITGB8, NTRK2, PTPRZ1, SLC12A7 and SLC1A3). For three genes (CLCN3, THBD and TNC), microarray and qRT-PCR analyses gave inconsistent results. Overall, our results show a statistically significant correlation between protein and mRNA expression levels for the cell surface proteins up-regulated in GBM tumors. We clearly confirm overexpression of 25 cell surface proteins in GBM tumors compared to astrocytes and NPCs, as the result from microarray and qRT-PCR analyses were overall in accordance with those of our differentially expressed glycoproteomic analysis. Thus, the genes showing the highest expression levels in GBM tumors compared to astrocytes, at both mRNA and protein levels, include SLC1A3, PTPRZ1, GPR56, NTRK2, IL13RA2, NRCAM, AGT and CLU. It’s interesting to note that our proteome and transcriptome results also indicated that the expression of EGFR, the most commonly altered gene in GBM with overexpression and/or mutation occurring in at least 50% of cases [46], was not significantly different between the tumor group and the astrocyte group. Our results are consistent with previous findings showing a loss of EGFR amplification in primary cultured gliomas [34], [35], [47], [48].

Bottom Line:
In this study, we used the bioorthogonal chemical reporter strategy (BOCR) in combination with label-free quantitative mass spectrometry (LFQ-MS) to characterize and accurately quantify the individual cell surface sialoproteome in human GBM tissues, in fetal, adult human astrocytes, and in human neural progenitor cells (NPCs).We identified and quantified a total of 843 proteins, including 801 glycoproteins.Among the 843 proteins, 606 (72%) are known cell surface or secreted glycoproteins, including 156 CD-antigens, all major classes of cell surface receptor proteins, transporters, and adhesion proteins.

ABSTRACTGlioblastoma multiform (GBM) remains clinical indication with significant "unmet medical need". Innovative new therapy to eliminate residual tumor cells and prevent tumor recurrences is critically needed for this deadly disease. A major challenge of GBM research has been the identification of novel molecular therapeutic targets and accurate diagnostic/prognostic biomarkers. Many of the current clinical therapeutic targets of immunotoxins and ligand-directed toxins for high-grade glioma (HGG) cells are surface sialylated glycoproteins. Therefore, methods that systematically and quantitatively analyze cell surface sialoglycoproteins in human clinical tumor samples would be useful for the identification of potential diagnostic markers and therapeutic targets for malignant gliomas. In this study, we used the bioorthogonal chemical reporter strategy (BOCR) in combination with label-free quantitative mass spectrometry (LFQ-MS) to characterize and accurately quantify the individual cell surface sialoproteome in human GBM tissues, in fetal, adult human astrocytes, and in human neural progenitor cells (NPCs). We identified and quantified a total of 843 proteins, including 801 glycoproteins. Among the 843 proteins, 606 (72%) are known cell surface or secreted glycoproteins, including 156 CD-antigens, all major classes of cell surface receptor proteins, transporters, and adhesion proteins. Our findings identified several known as well as new cell surface antigens whose expression is predominantly restricted to human GBM tumors as confirmed by microarray transcription profiling, quantitative RT-PCR and immunohistochemical staining. This report presents the comprehensive identification of new biomarkers and therapeutic targets for the treatment of malignant gliomas using quantitative sialoglycoproteomics with clinically relevant, patient derived primary glioma cells.